The Binding Properties of Glycosylated and Non- Glycosylated Tim-3 Molecules on CD4＋CD25＋ T Cells
Lee, MJ; Heo, YM; Hong, SH; Kim, K; Park, S
Immune network, 9(2):58-63, 2009
Background: T cell immunoglobulin and mucin domain containing 3 protein (Tim-3) expressed on terminally differentiated Th1 cells plays a suppressive role in Th1-mediated immune responses. Recently, it has been shown that N-glycosylation affects the binding activity of the Tim-3-Ig fusion protein to its ligand, galectin-9, but the binding properties of non-glycosylated Tim-3 on CD4＋CD25＋ T cells has not been fully examined. In this study, we produced recombinant Tim- 3-Ig fusion proteins in different cellular sources and its N-glycosylation mutant forms to evaluate their binding activities to CD4＋CD25＋ T cells.
Methods: We isolated and cloned Tim- 3 cDNA from BALB/C mouse splenocytes. Then, we constructed a mammalian expression vector and a prokaryotic expression vector for the Tim-3-Ig fusion protein. Using a site directed mutagenesis method, plasmid vectors for Tim-3-Ig N-glycosylation mutant expression were produced. The recombinant protein was purified by protein A sepharose column chromatography. The binding activity of Tim-3-Ig fusion protein to CD4＋CD25＋ T cells was analyzed using flow cytometry.
Results: We found that the nonglycosylated Tim- 3-Ig fusion proteins expressed in bacteria bound to CD4＋ CD25＋ T cells similarly to the glycosylated Tim-3-Ig protein produced in CHO cells. Further, three N-glycosylation mutant forms (N53Q, N100Q, N53/100Q) of Tim-3-Ig showed similar binding activities to those of wild type glycosylated Tim-3-Ig.
Conclusion: Our results suggest that N-glycosylation of Tim-3 may not affect its binding activity to ligands expressed on CD4＋CD25＋ T cells.
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